摘要
目的重组表达肠出血性大肠杆菌（EHEC）O157：H7的EspA与Stx2B融合蛋白，并对其生物学活性进行初步研究。方法采用PCR技术从EHEC O157：H7基因组中扩增espA基因及stx2B基因，两基因通过连接子（1inker）相连，T／A法克隆，克隆至pET-28a（＋）表达载体上，转化宿主细胞E.coli BL21（DE3），IPTG诱导表达，SDS—PAGE检测其表达量及表达形式，免疫印迹分析免疫反应性。用纯化的融合蛋白免疫BALB／c小鼠，检测其免疫原性，然后对免疫小鼠进行O157活菌攻毒试验和O157超声上清致死保护试验。结果构建的espa—stx2B融合基因片段的测序结果与理论预测值完全一致。融合蛋白在工程菌中以包涵体的形式表达，表达量约40％。免疫印迹显示融合蛋白能分别与EspA和Stx2B抗体发生抗原抗体反应。免疫小鼠其特异性抗体阳转率达100％，EspA和Stx2B抗体的几何平均滴度（GMT）分别增长了124．30倍和58．49倍。免疫小鼠O157活菌攻毒保护试验免疫后排菌时间和排菌量与对照组相比并没有明显改变，O157菌超声上清致死保护试验免疫组存活率为10／15，对照组全部死亡。结论在E．coli中高效表达了EspA—Stx2B融合蛋白，此融合蛋白具有良好的免疫原性和免疫反应性，融合蛋白免疫小鼠并不能降低细菌在小鼠胃肠道的黏附与定植，但却起到明显的免疫保护作用，保护率为66．7％（10／15）。
Objective To clone the gene encoding protein of EspA and Stx2B from EHEC O157：H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a （ ＋ ）-espA- stx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a（ ＋ ） vector. The vector was then transferred to the host cells E. coli BL21 strain （DE3）. Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fu- sion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of O157 in mice. In the test of death of BALB/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B protein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immnnogenicity. EspA-Stx2B could not decrease bacterial number attached to the
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第3期258-262,共5页
Chinese Journal of Microbiology and Immunology
基金
军队杰出人才基金资助项目（04J009）
